RATIONALE: Standardization of allergen exposure measurements is important to ensure reproducibili... more RATIONALE: Standardization of allergen exposure measurements is important to ensure reproducibility of results obtained by different laboratories. We evaluated reproducibility and inter-laboratory variability of a Multiplex Array for Indoor Allergens (MARIA), including Der p 1, Der f 1, Mite Group 2, Fel d 1, Can f 1, Rat n 1, Mus m 1 and Bla g 2. METHODS: For evaluation of the 8-plex MARIA, we used a multi-center ring trial. Ten laboratories across the US and Europe were recruited and trained to use the technology. All reagents required for the trial, as well as aliquots of an identical set of 151 dust extract samples, were sent to each of the 10 participating centers and analyzed by each laboratory on three separate occasions at three dilutions. A hierarchical model was applied to the nested data structure (repeat nested within laboratories, laboratories nested within samples). RESULTS: Complete results for 3 of the 10 participating laboratories were available at time of submission. The current results are based on more than 10,000 individual allergen measurements. More than 36,000 tests will eventually be completed. Allergens levels covered a wide range from below detection limit to greater than 100ug/g (Mus m 1 < 35ug/g). Results were highly reproducible within as well as between the three laboratories, with correlation coefficients generally greater than 0.95. CONCLUSIONS: The data indicate that the MARIA produces results that are reproducible within and between laboratories, which will improve standardization of allergen exposure assessment.
RATIONALE: Standardization of allergen exposure measurements is important to ensure reproducibili... more RATIONALE: Standardization of allergen exposure measurements is important to ensure reproducibility of results obtained by different laboratories. We evaluated reproducibility and inter-laboratory variability of a Multiplex Array for Indoor Allergens (MARIA), including Der p 1, Der f 1, Mite Group 2, Fel d 1, Can f 1, Rat n 1, Mus m 1 and Bla g 2. METHODS: For evaluation of the 8-plex MARIA, we used a multi-center ring trial. Ten laboratories across the US and Europe were recruited and trained to use the technology. All reagents required for the trial, as well as aliquots of an identical set of 151 dust extract samples, were sent to each of the 10 participating centers and analyzed by each laboratory on three separate occasions at three dilutions. A hierarchical model was applied to the nested data structure (repeat nested within laboratories, laboratories nested within samples). RESULTS: Complete results for 3 of the 10 participating laboratories were available at time of submission. The current results are based on more than 10,000 individual allergen measurements. More than 36,000 tests will eventually be completed. Allergens levels covered a wide range from below detection limit to greater than 100ug/g (Mus m 1 < 35ug/g). Results were highly reproducible within as well as between the three laboratories, with correlation coefficients generally greater than 0.95. CONCLUSIONS: The data indicate that the MARIA produces results that are reproducible within and between laboratories, which will improve standardization of allergen exposure assessment.
RATIONALE: Standardization of allergen exposure measurements is important to ensure reproducibili... more RATIONALE: Standardization of allergen exposure measurements is important to ensure reproducibility of results obtained by different laboratories. We evaluated reproducibility and inter-laboratory variability of a Multiplex Array for Indoor Allergens (MARIA), including Der p 1, Der f 1, Mite Group 2, Fel d 1, Can f 1, Rat n 1, Mus m 1 and Bla g 2. METHODS: For evaluation of the 8-plex MARIA, we used a multi-center ring trial. Ten laboratories across the US and Europe were recruited and trained to use the technology. All reagents required for the trial, as well as aliquots of an identical set of 151 dust extract samples, were sent to each of the 10 participating centers and analyzed by each laboratory on three separate occasions at three dilutions. A hierarchical model was applied to the nested data structure (repeat nested within laboratories, laboratories nested within samples). RESULTS: Complete results for 3 of the 10 participating laboratories were available at time of submission. The current results are based on more than 10,000 individual allergen measurements. More than 36,000 tests will eventually be completed. Allergens levels covered a wide range from below detection limit to greater than 100ug/g (Mus m 1 < 35ug/g). Results were highly reproducible within as well as between the three laboratories, with correlation coefficients generally greater than 0.95. CONCLUSIONS: The data indicate that the MARIA produces results that are reproducible within and between laboratories, which will improve standardization of allergen exposure assessment.
Current diagnostics for grass pollen allergy are composed of mixtures of pollen of different gras... more Current diagnostics for grass pollen allergy are composed of mixtures of pollen of different grass species. Their complex composition hampers accurate standardization. The aim of the study was to investigate whether mixtures of grass pollen extracts can be replaced by a single pollen species and whether a single pollen species can be replaced by a limited number of purified natural or recombinant major allergens. Sera (n = 800) were selected on the basis of a general suspicion for inhalant allergy and tested in a RAST for IgE reactivity with pollen from 17 different grass species. Cross-reactivity of IgE responses was studied by means of RAST inhibition. Sera with positive test results for grass pollen were tested in a RAST for natural Lol p 1 and Lol p 5 and recombinant Phl p 1 and Phl p 5. Specific IgE antibodies against one or more of the 17 pollen species were detected in 209 of 800 sera (26.1%). The highest responses were observed against Poa pratensis followed by Festuca rubra, Phleum pratense, and Dactylis glomerata. IgE responses were clearly lower (approximately by a factor of 5) against only three species (Phragmites communis, Cynodon dactylon, and Zea mays). With the exception of a few low-responder sera, no sera were found to have negative test results to the high responder species and positive results to any of the other species. Sera with positive test results for grass pollen (n = 154) were tested with purified Lol p 1 and Lol p 5. IgE anti-Lol p 1 and Lol p 5 accounted for an average of 81% +/- 7% of total anti-grass pollen IgE. For 14 sera (all with low anti-grass pollen IgE titers), a RAST with purified allergens resulted in a false-negative diagnosis for grass pollen allergy. With recombinant Phl p 1 and Phl p 5, the mean IgE reactivity was 57% +/- 6% of the anti-grass pollen IgE response (n = 141), with 13 false-negative results. One grass species is sufficient for in vitro diagnosis of grass pollen allergy. With purified natural Lol p 1 and Lol p 5, greater than 90% of grass-positive sera is detected. Around 80% of the IgE response to grass pollen is directed to these major allergens. Recombinant allergens, produced in Escherichia coli, did not equal the IgE-binding capacity of their natural counterparts.
Current diagnostics for grass pollen allergy are composed of mixtures of pollen of different gras... more Current diagnostics for grass pollen allergy are composed of mixtures of pollen of different grass species. Their complex composition hampers accurate standardization. The aim of the study was to investigate whether mixtures of grass pollen extracts can be replaced by a single pollen species and whether a single pollen species can be replaced by a limited number of purified natural or recombinant major allergens. Sera (n = 800) were selected on the basis of a general suspicion for inhalant allergy and tested in a RAST for IgE reactivity with pollen from 17 different grass species. Cross-reactivity of IgE responses was studied by means of RAST inhibition. Sera with positive test results for grass pollen were tested in a RAST for natural Lol p 1 and Lol p 5 and recombinant Phl p 1 and Phl p 5. Specific IgE antibodies against one or more of the 17 pollen species were detected in 209 of 800 sera (26.1%). The highest responses were observed against Poa pratensis followed by Festuca rubra, Phleum pratense, and Dactylis glomerata. IgE responses were clearly lower (approximately by a factor of 5) against only three species (Phragmites communis, Cynodon dactylon, and Zea mays). With the exception of a few low-responder sera, no sera were found to have negative test results to the high responder species and positive results to any of the other species. Sera with positive test results for grass pollen (n = 154) were tested with purified Lol p 1 and Lol p 5. IgE anti-Lol p 1 and Lol p 5 accounted for an average of 81% +/- 7% of total anti-grass pollen IgE. For 14 sera (all with low anti-grass pollen IgE titers), a RAST with purified allergens resulted in a false-negative diagnosis for grass pollen allergy. With recombinant Phl p 1 and Phl p 5, the mean IgE reactivity was 57% +/- 6% of the anti-grass pollen IgE response (n = 141), with 13 false-negative results. One grass species is sufficient for in vitro diagnosis of grass pollen allergy. With purified natural Lol p 1 and Lol p 5, greater than 90% of grass-positive sera is detected. Around 80% of the IgE response to grass pollen is directed to these major allergens. Recombinant allergens, produced in Escherichia coli, did not equal the IgE-binding capacity of their natural counterparts.
Current diagnostics for grass pollen allergy are composed of mixtures of pollen of different gras... more Current diagnostics for grass pollen allergy are composed of mixtures of pollen of different grass species. Their complex composition hampers accurate standardization. The aim of the study was to investigate whether mixtures of grass pollen extracts can be replaced by a single pollen species and whether a single pollen species can be replaced by a limited number of purified natural or recombinant major allergens. Sera (n = 800) were selected on the basis of a general suspicion for inhalant allergy and tested in a RAST for IgE reactivity with pollen from 17 different grass species. Cross-reactivity of IgE responses was studied by means of RAST inhibition. Sera with positive test results for grass pollen were tested in a RAST for natural Lol p 1 and Lol p 5 and recombinant Phl p 1 and Phl p 5. Specific IgE antibodies against one or more of the 17 pollen species were detected in 209 of 800 sera (26.1%). The highest responses were observed against Poa pratensis followed by Festuca rubra, Phleum pratense, and Dactylis glomerata. IgE responses were clearly lower (approximately by a factor of 5) against only three species (Phragmites communis, Cynodon dactylon, and Zea mays). With the exception of a few low-responder sera, no sera were found to have negative test results to the high responder species and positive results to any of the other species. Sera with positive test results for grass pollen (n = 154) were tested with purified Lol p 1 and Lol p 5. IgE anti-Lol p 1 and Lol p 5 accounted for an average of 81% +/- 7% of total anti-grass pollen IgE. For 14 sera (all with low anti-grass pollen IgE titers), a RAST with purified allergens resulted in a false-negative diagnosis for grass pollen allergy. With recombinant Phl p 1 and Phl p 5, the mean IgE reactivity was 57% +/- 6% of the anti-grass pollen IgE response (n = 141), with 13 false-negative results. One grass species is sufficient for in vitro diagnosis of grass pollen allergy. With purified natural Lol p 1 and Lol p 5, greater than 90% of grass-positive sera is detected. Around 80% of the IgE response to grass pollen is directed to these major allergens. Recombinant allergens, produced in Escherichia coli, did not equal the IgE-binding capacity of their natural counterparts.
International Archives of Allergy and Immunology, 1996
Background: In order to investigate, whether atopic and nonatopic children show differences in th... more Background: In order to investigate, whether atopic and nonatopic children show differences in their specific IgE and IgG4 immune responses to tetanus (T) and diphtheria (D) antigens, we studied 538 children who had been followed from birth on and from whom records had been kept of all immunizations. Methods: The prevalence of eczema and asthma was registered at regular intervals
International Archives of Allergy and Immunology, 1996
Background: In order to investigate, whether atopic and nonatopic children show differences in th... more Background: In order to investigate, whether atopic and nonatopic children show differences in their specific IgE and IgG4 immune responses to tetanus (T) and diphtheria (D) antigens, we studied 538 children who had been followed from birth on and from whom records had been kept of all immunizations. Methods: The prevalence of eczema and asthma was registered at regular intervals
Regulatory toxicology and pharmacology : RTP, 2004
Rationale. Evaluation of the potential allergenicity of proteins derived from genetically modifie... more Rationale. Evaluation of the potential allergenicity of proteins derived from genetically modified foods has involved a weight of evidence approach that incorporates an evaluation of protein digestibility in pepsin. Currently, there is no standardized protocol to assess the digestibility of proteins using simulated gastric fluid. Potential variations in assay parameters include: pH, pepsin purity, pepsin to target protein ratio, target protein purity, and method of detection. The objective was to assess the digestibility of a common set of proteins in nine independent laboratories to determine the reproducibility of the assay when performed using a common protocol. Methods. A single lot of each test protein and pepsin was obtained and distributed to each laboratory. The test proteins consisted of Ara h 2 (a peanut conglutin-like protein), beta-lactoglobulin, bovine serum albumin, concanavalin A, horseradish peroxidase, ovalbumin, ovomucoid, phosphinothricin acetyltransferase, ribulo...
Evaluation of the potential allergenicity of food proteins derived from genetically modified crop... more Evaluation of the potential allergenicity of food proteins derived from genetically modified crop plants has conventionally involved the use of a decision tree that incorporates an evaluation of protein digestibility in pepsin. Currently, there is no commonly accepted protocol to assess the digestibility of proteins using simulated gastric fluid, although it is a requirement for regulatory submission. Potential variations in assay parameters include: pH, pepsin purity, pepsin-to-target protein ratio, target protein purity, and method of detection.
International Archives of Allergy and Immunology, 2005
The first genetically modified (GM) crops approved for food use (tomato and soybean) were evaluat... more The first genetically modified (GM) crops approved for food use (tomato and soybean) were evaluated for safety by the United States Food and Drug Administration prior to commercial production. Among other factors, those products and all additional GM crops that have been grown commercially have been evaluated for potential increases in allergenic properties using methods that are consistent with the
Allergy: European Journal of Allergy and Clinical Immunology, 2013
Background: Complaints of 'food allergy' are increasing. Standardized surveys of IgE sensitizatio... more Background: Complaints of 'food allergy' are increasing. Standardized surveys of IgE sensitization to foods are still uncommon and multicountry surveys are rare. We have assessed IgE sensitization to food-associated allergens in different regions of Europe using a common protocol. Methods: Participants from general populations aged 20-54 years in eight European centres (Zurich, Madrid, Utrecht, Lodz, Sophia, Athens, Reykjavik and Vilnius) were asked whether they had allergic symptoms associated with specific foods. Weighted samples of those with and without allergic symptoms then completed a longer questionnaire and donated serum for IgE analysis by Immuno-CAP for 24 foods, 6 aeroallergens and, by allergen microarray, for 48 individual food proteins. Results: The prevalence of IgE sensitization to foods ranged from 23.6% to 6.6%. The least common IgE sensitizations were to fish (0.2%), milk (0.8%) and egg (0.9%), and the most common were to hazelnut (9.3%), peach (7.9%) and apple (6.5%). The order of prevalence of IgE sensitization against different foods was similar in each centre and correlated with the prevalence of the pollen-associated allergens Bet v 1 and Bet v 2 (r = 0.86). IgE sensitization to plant allergen components unrelated to pollen allergens was more evenly distributed and independent of pollen IgE sensitization (r = À0.10). The most common foods containing allergens not cross-reacting with pollens were sesame, shrimp and hazelnut. Discussion: IgE sensitization to foods is common, but varies widely and is predominantly related to IgE sensitization to pollen allergens. IgE sensitization to food allergens not cross-reacting with pollens is rare and more evenly distributed.
International Archives of Allergy and Immunology, 2004
Recombinant proteins from Pichia pastoris need to be fully evaluated before used as diagnostic to... more Recombinant proteins from Pichia pastoris need to be fully evaluated before used as diagnostic tools. The objective of this study was to investigate whether glycosylation by P. pastoris interferes with the specificity of diagnostic tests. An autoantigen involved in Wegener&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;s disease (protease 3) and 2 major inhalant allergens from grass pollen (Dac g 5) and house dust mite (Der p 1) were produced as recombinant molecules in P. pastoris. O-linked glycans on Dac g 5 were characterized by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The immune reactivity of the recombinant proteins was compared to that of their natural counterparts by ELISA and a radio-allergosorbent test (RAST) as well as by ELISA and RAST inhibition. In contrast to the non-glycosylated natural allergen, recombinant Dac g 5 was shown to carry at least 2 small mannose-containing O-glycans. We showed that both these O-glycans and the N-linked glycans on recombinant protease 3 and recombinant Der p 1 were recognized in ELISA by IgG antibodies in sera of healthy individuals. These IgG responses were closely correlated. The natural autoantigen and allergens were not recognized by IgG antibodies from healthy subjects. The carbohydrate nature of the epitopes recognized by IgG on the recombinant proteins was confirmed by inhibition studies with mannose and yeast mannan. IgE recognition of yeast glycans was observed in 2 out of 9 positive sera from patients with allergic bronchopulmonary aspergillosis. Production of recombinant molecules in yeast (or moulds) can introduce IgG-binding glycans that negatively affect the specificity of diagnostic tests.
RATIONALE: Standardization of allergen exposure measurements is important to ensure reproducibili... more RATIONALE: Standardization of allergen exposure measurements is important to ensure reproducibility of results obtained by different laboratories. We evaluated reproducibility and inter-laboratory variability of a Multiplex Array for Indoor Allergens (MARIA), including Der p 1, Der f 1, Mite Group 2, Fel d 1, Can f 1, Rat n 1, Mus m 1 and Bla g 2. METHODS: For evaluation of the 8-plex MARIA, we used a multi-center ring trial. Ten laboratories across the US and Europe were recruited and trained to use the technology. All reagents required for the trial, as well as aliquots of an identical set of 151 dust extract samples, were sent to each of the 10 participating centers and analyzed by each laboratory on three separate occasions at three dilutions. A hierarchical model was applied to the nested data structure (repeat nested within laboratories, laboratories nested within samples). RESULTS: Complete results for 3 of the 10 participating laboratories were available at time of submission. The current results are based on more than 10,000 individual allergen measurements. More than 36,000 tests will eventually be completed. Allergens levels covered a wide range from below detection limit to greater than 100ug/g (Mus m 1 < 35ug/g). Results were highly reproducible within as well as between the three laboratories, with correlation coefficients generally greater than 0.95. CONCLUSIONS: The data indicate that the MARIA produces results that are reproducible within and between laboratories, which will improve standardization of allergen exposure assessment.
RATIONALE: Standardization of allergen exposure measurements is important to ensure reproducibili... more RATIONALE: Standardization of allergen exposure measurements is important to ensure reproducibility of results obtained by different laboratories. We evaluated reproducibility and inter-laboratory variability of a Multiplex Array for Indoor Allergens (MARIA), including Der p 1, Der f 1, Mite Group 2, Fel d 1, Can f 1, Rat n 1, Mus m 1 and Bla g 2. METHODS: For evaluation of the 8-plex MARIA, we used a multi-center ring trial. Ten laboratories across the US and Europe were recruited and trained to use the technology. All reagents required for the trial, as well as aliquots of an identical set of 151 dust extract samples, were sent to each of the 10 participating centers and analyzed by each laboratory on three separate occasions at three dilutions. A hierarchical model was applied to the nested data structure (repeat nested within laboratories, laboratories nested within samples). RESULTS: Complete results for 3 of the 10 participating laboratories were available at time of submission. The current results are based on more than 10,000 individual allergen measurements. More than 36,000 tests will eventually be completed. Allergens levels covered a wide range from below detection limit to greater than 100ug/g (Mus m 1 < 35ug/g). Results were highly reproducible within as well as between the three laboratories, with correlation coefficients generally greater than 0.95. CONCLUSIONS: The data indicate that the MARIA produces results that are reproducible within and between laboratories, which will improve standardization of allergen exposure assessment.
RATIONALE: Standardization of allergen exposure measurements is important to ensure reproducibili... more RATIONALE: Standardization of allergen exposure measurements is important to ensure reproducibility of results obtained by different laboratories. We evaluated reproducibility and inter-laboratory variability of a Multiplex Array for Indoor Allergens (MARIA), including Der p 1, Der f 1, Mite Group 2, Fel d 1, Can f 1, Rat n 1, Mus m 1 and Bla g 2. METHODS: For evaluation of the 8-plex MARIA, we used a multi-center ring trial. Ten laboratories across the US and Europe were recruited and trained to use the technology. All reagents required for the trial, as well as aliquots of an identical set of 151 dust extract samples, were sent to each of the 10 participating centers and analyzed by each laboratory on three separate occasions at three dilutions. A hierarchical model was applied to the nested data structure (repeat nested within laboratories, laboratories nested within samples). RESULTS: Complete results for 3 of the 10 participating laboratories were available at time of submission. The current results are based on more than 10,000 individual allergen measurements. More than 36,000 tests will eventually be completed. Allergens levels covered a wide range from below detection limit to greater than 100ug/g (Mus m 1 < 35ug/g). Results were highly reproducible within as well as between the three laboratories, with correlation coefficients generally greater than 0.95. CONCLUSIONS: The data indicate that the MARIA produces results that are reproducible within and between laboratories, which will improve standardization of allergen exposure assessment.
Current diagnostics for grass pollen allergy are composed of mixtures of pollen of different gras... more Current diagnostics for grass pollen allergy are composed of mixtures of pollen of different grass species. Their complex composition hampers accurate standardization. The aim of the study was to investigate whether mixtures of grass pollen extracts can be replaced by a single pollen species and whether a single pollen species can be replaced by a limited number of purified natural or recombinant major allergens. Sera (n = 800) were selected on the basis of a general suspicion for inhalant allergy and tested in a RAST for IgE reactivity with pollen from 17 different grass species. Cross-reactivity of IgE responses was studied by means of RAST inhibition. Sera with positive test results for grass pollen were tested in a RAST for natural Lol p 1 and Lol p 5 and recombinant Phl p 1 and Phl p 5. Specific IgE antibodies against one or more of the 17 pollen species were detected in 209 of 800 sera (26.1%). The highest responses were observed against Poa pratensis followed by Festuca rubra, Phleum pratense, and Dactylis glomerata. IgE responses were clearly lower (approximately by a factor of 5) against only three species (Phragmites communis, Cynodon dactylon, and Zea mays). With the exception of a few low-responder sera, no sera were found to have negative test results to the high responder species and positive results to any of the other species. Sera with positive test results for grass pollen (n = 154) were tested with purified Lol p 1 and Lol p 5. IgE anti-Lol p 1 and Lol p 5 accounted for an average of 81% +/- 7% of total anti-grass pollen IgE. For 14 sera (all with low anti-grass pollen IgE titers), a RAST with purified allergens resulted in a false-negative diagnosis for grass pollen allergy. With recombinant Phl p 1 and Phl p 5, the mean IgE reactivity was 57% +/- 6% of the anti-grass pollen IgE response (n = 141), with 13 false-negative results. One grass species is sufficient for in vitro diagnosis of grass pollen allergy. With purified natural Lol p 1 and Lol p 5, greater than 90% of grass-positive sera is detected. Around 80% of the IgE response to grass pollen is directed to these major allergens. Recombinant allergens, produced in Escherichia coli, did not equal the IgE-binding capacity of their natural counterparts.
Current diagnostics for grass pollen allergy are composed of mixtures of pollen of different gras... more Current diagnostics for grass pollen allergy are composed of mixtures of pollen of different grass species. Their complex composition hampers accurate standardization. The aim of the study was to investigate whether mixtures of grass pollen extracts can be replaced by a single pollen species and whether a single pollen species can be replaced by a limited number of purified natural or recombinant major allergens. Sera (n = 800) were selected on the basis of a general suspicion for inhalant allergy and tested in a RAST for IgE reactivity with pollen from 17 different grass species. Cross-reactivity of IgE responses was studied by means of RAST inhibition. Sera with positive test results for grass pollen were tested in a RAST for natural Lol p 1 and Lol p 5 and recombinant Phl p 1 and Phl p 5. Specific IgE antibodies against one or more of the 17 pollen species were detected in 209 of 800 sera (26.1%). The highest responses were observed against Poa pratensis followed by Festuca rubra, Phleum pratense, and Dactylis glomerata. IgE responses were clearly lower (approximately by a factor of 5) against only three species (Phragmites communis, Cynodon dactylon, and Zea mays). With the exception of a few low-responder sera, no sera were found to have negative test results to the high responder species and positive results to any of the other species. Sera with positive test results for grass pollen (n = 154) were tested with purified Lol p 1 and Lol p 5. IgE anti-Lol p 1 and Lol p 5 accounted for an average of 81% +/- 7% of total anti-grass pollen IgE. For 14 sera (all with low anti-grass pollen IgE titers), a RAST with purified allergens resulted in a false-negative diagnosis for grass pollen allergy. With recombinant Phl p 1 and Phl p 5, the mean IgE reactivity was 57% +/- 6% of the anti-grass pollen IgE response (n = 141), with 13 false-negative results. One grass species is sufficient for in vitro diagnosis of grass pollen allergy. With purified natural Lol p 1 and Lol p 5, greater than 90% of grass-positive sera is detected. Around 80% of the IgE response to grass pollen is directed to these major allergens. Recombinant allergens, produced in Escherichia coli, did not equal the IgE-binding capacity of their natural counterparts.
Current diagnostics for grass pollen allergy are composed of mixtures of pollen of different gras... more Current diagnostics for grass pollen allergy are composed of mixtures of pollen of different grass species. Their complex composition hampers accurate standardization. The aim of the study was to investigate whether mixtures of grass pollen extracts can be replaced by a single pollen species and whether a single pollen species can be replaced by a limited number of purified natural or recombinant major allergens. Sera (n = 800) were selected on the basis of a general suspicion for inhalant allergy and tested in a RAST for IgE reactivity with pollen from 17 different grass species. Cross-reactivity of IgE responses was studied by means of RAST inhibition. Sera with positive test results for grass pollen were tested in a RAST for natural Lol p 1 and Lol p 5 and recombinant Phl p 1 and Phl p 5. Specific IgE antibodies against one or more of the 17 pollen species were detected in 209 of 800 sera (26.1%). The highest responses were observed against Poa pratensis followed by Festuca rubra, Phleum pratense, and Dactylis glomerata. IgE responses were clearly lower (approximately by a factor of 5) against only three species (Phragmites communis, Cynodon dactylon, and Zea mays). With the exception of a few low-responder sera, no sera were found to have negative test results to the high responder species and positive results to any of the other species. Sera with positive test results for grass pollen (n = 154) were tested with purified Lol p 1 and Lol p 5. IgE anti-Lol p 1 and Lol p 5 accounted for an average of 81% +/- 7% of total anti-grass pollen IgE. For 14 sera (all with low anti-grass pollen IgE titers), a RAST with purified allergens resulted in a false-negative diagnosis for grass pollen allergy. With recombinant Phl p 1 and Phl p 5, the mean IgE reactivity was 57% +/- 6% of the anti-grass pollen IgE response (n = 141), with 13 false-negative results. One grass species is sufficient for in vitro diagnosis of grass pollen allergy. With purified natural Lol p 1 and Lol p 5, greater than 90% of grass-positive sera is detected. Around 80% of the IgE response to grass pollen is directed to these major allergens. Recombinant allergens, produced in Escherichia coli, did not equal the IgE-binding capacity of their natural counterparts.
International Archives of Allergy and Immunology, 1996
Background: In order to investigate, whether atopic and nonatopic children show differences in th... more Background: In order to investigate, whether atopic and nonatopic children show differences in their specific IgE and IgG4 immune responses to tetanus (T) and diphtheria (D) antigens, we studied 538 children who had been followed from birth on and from whom records had been kept of all immunizations. Methods: The prevalence of eczema and asthma was registered at regular intervals
International Archives of Allergy and Immunology, 1996
Background: In order to investigate, whether atopic and nonatopic children show differences in th... more Background: In order to investigate, whether atopic and nonatopic children show differences in their specific IgE and IgG4 immune responses to tetanus (T) and diphtheria (D) antigens, we studied 538 children who had been followed from birth on and from whom records had been kept of all immunizations. Methods: The prevalence of eczema and asthma was registered at regular intervals
Regulatory toxicology and pharmacology : RTP, 2004
Rationale. Evaluation of the potential allergenicity of proteins derived from genetically modifie... more Rationale. Evaluation of the potential allergenicity of proteins derived from genetically modified foods has involved a weight of evidence approach that incorporates an evaluation of protein digestibility in pepsin. Currently, there is no standardized protocol to assess the digestibility of proteins using simulated gastric fluid. Potential variations in assay parameters include: pH, pepsin purity, pepsin to target protein ratio, target protein purity, and method of detection. The objective was to assess the digestibility of a common set of proteins in nine independent laboratories to determine the reproducibility of the assay when performed using a common protocol. Methods. A single lot of each test protein and pepsin was obtained and distributed to each laboratory. The test proteins consisted of Ara h 2 (a peanut conglutin-like protein), beta-lactoglobulin, bovine serum albumin, concanavalin A, horseradish peroxidase, ovalbumin, ovomucoid, phosphinothricin acetyltransferase, ribulo...
Evaluation of the potential allergenicity of food proteins derived from genetically modified crop... more Evaluation of the potential allergenicity of food proteins derived from genetically modified crop plants has conventionally involved the use of a decision tree that incorporates an evaluation of protein digestibility in pepsin. Currently, there is no commonly accepted protocol to assess the digestibility of proteins using simulated gastric fluid, although it is a requirement for regulatory submission. Potential variations in assay parameters include: pH, pepsin purity, pepsin-to-target protein ratio, target protein purity, and method of detection.
International Archives of Allergy and Immunology, 2005
The first genetically modified (GM) crops approved for food use (tomato and soybean) were evaluat... more The first genetically modified (GM) crops approved for food use (tomato and soybean) were evaluated for safety by the United States Food and Drug Administration prior to commercial production. Among other factors, those products and all additional GM crops that have been grown commercially have been evaluated for potential increases in allergenic properties using methods that are consistent with the
Allergy: European Journal of Allergy and Clinical Immunology, 2013
Background: Complaints of 'food allergy' are increasing. Standardized surveys of IgE sensitizatio... more Background: Complaints of 'food allergy' are increasing. Standardized surveys of IgE sensitization to foods are still uncommon and multicountry surveys are rare. We have assessed IgE sensitization to food-associated allergens in different regions of Europe using a common protocol. Methods: Participants from general populations aged 20-54 years in eight European centres (Zurich, Madrid, Utrecht, Lodz, Sophia, Athens, Reykjavik and Vilnius) were asked whether they had allergic symptoms associated with specific foods. Weighted samples of those with and without allergic symptoms then completed a longer questionnaire and donated serum for IgE analysis by Immuno-CAP for 24 foods, 6 aeroallergens and, by allergen microarray, for 48 individual food proteins. Results: The prevalence of IgE sensitization to foods ranged from 23.6% to 6.6%. The least common IgE sensitizations were to fish (0.2%), milk (0.8%) and egg (0.9%), and the most common were to hazelnut (9.3%), peach (7.9%) and apple (6.5%). The order of prevalence of IgE sensitization against different foods was similar in each centre and correlated with the prevalence of the pollen-associated allergens Bet v 1 and Bet v 2 (r = 0.86). IgE sensitization to plant allergen components unrelated to pollen allergens was more evenly distributed and independent of pollen IgE sensitization (r = À0.10). The most common foods containing allergens not cross-reacting with pollens were sesame, shrimp and hazelnut. Discussion: IgE sensitization to foods is common, but varies widely and is predominantly related to IgE sensitization to pollen allergens. IgE sensitization to food allergens not cross-reacting with pollens is rare and more evenly distributed.
International Archives of Allergy and Immunology, 2004
Recombinant proteins from Pichia pastoris need to be fully evaluated before used as diagnostic to... more Recombinant proteins from Pichia pastoris need to be fully evaluated before used as diagnostic tools. The objective of this study was to investigate whether glycosylation by P. pastoris interferes with the specificity of diagnostic tests. An autoantigen involved in Wegener&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;s disease (protease 3) and 2 major inhalant allergens from grass pollen (Dac g 5) and house dust mite (Der p 1) were produced as recombinant molecules in P. pastoris. O-linked glycans on Dac g 5 were characterized by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The immune reactivity of the recombinant proteins was compared to that of their natural counterparts by ELISA and a radio-allergosorbent test (RAST) as well as by ELISA and RAST inhibition. In contrast to the non-glycosylated natural allergen, recombinant Dac g 5 was shown to carry at least 2 small mannose-containing O-glycans. We showed that both these O-glycans and the N-linked glycans on recombinant protease 3 and recombinant Der p 1 were recognized in ELISA by IgG antibodies in sera of healthy individuals. These IgG responses were closely correlated. The natural autoantigen and allergens were not recognized by IgG antibodies from healthy subjects. The carbohydrate nature of the epitopes recognized by IgG on the recombinant proteins was confirmed by inhibition studies with mannose and yeast mannan. IgE recognition of yeast glycans was observed in 2 out of 9 positive sera from patients with allergic bronchopulmonary aspergillosis. Production of recombinant molecules in yeast (or moulds) can introduce IgG-binding glycans that negatively affect the specificity of diagnostic tests.
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Papers by R. Van Ree